Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the oxidative pentose phosphate cycle (OPPC). Glucose, a substrate for the OPPC, is required for OPPC mediated detoxification of oxidants/disulfides. Glucose is utilized as a substrate by oxidative pentose phosphate cycle to generate reductants. These reductants are utilized to maintain reduced glutathione homeostasis in mammalian cells when exposed to oxidants/disulfides. Glutathione is a tripeptide consisting of glycine, cysteine and glutamate. The reduced glutathione (GSH) is up to 100 folds higher than the oxidized GSH (GSSG) in mammalian cells under normal conditions.
Oxidative stress is presently quantified by measuring the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). Oxidized glutathione is the most commonly used biomarker in biomedical research. However, the various biochemical assays currently available require the preparation of tissue extracts and cannot be applied to humans because of their invasiveness. Indeed, while HPLC with electrochemical detection can monitor glutathione with better sensitivity than other biochemical assays, the method still requires tissue or cellular extracts. Furthermore, these assays may overestimate the extent of oxidative stress since depletion of GSH measured by biochemical assays may also include oxidation of GSH during lysis of cells and extract preparation. Moreover, none of these assays measures the function of GSH in live cells. In view of the foregoing, it is evident that there is a need for improved oxidative stress assays in live cells.